In the first session, subjects performed a graded exercise test to determine the cycling power output corresponding to VO2peak. The second session consisted of (a) pre-intervention assessment ; (b) exercise intervention (10 × 1-min of cycling at VO2peak power output); (c) post-intervention assessment, and (d) 30-min post-intervention evaluation. Significant post-exercise increases in corticotropin and cortisol plasma levels were observed whereas mood states decreased significantly at this assessment time-point. However, a significant increase in mood was found 30-min after exercise. Finally, significant relationships between increases in stress hormones concentrations and changes in mood states after intense exercise were observed. In conclusion, our HIIT-based intervention was feasible to deliver and acceptable to participants. A single bout of HIIT induced acute changes in mood states that seems to be associated with hypothalamic-pituitary-adrenal axis activation.This pilot study, conducted in advance of a future definitive randomized controlled trial, aimed to investigate the feasibility of using a HIIT-based intervention to induce neurophysiological stress responses that could be associated with possible changes in mood. Twenty-five active male college students with an average age of 21.7 ± 2.1 years, weight 72.6 ± 8.4 kg, height 177 ± 6.1 cm, and BMI: 23.1 ± 1.4 kg/m High-intensity interval training (HIIT) consists of short bursts of exercise followed by recovery periods . Thus, H2max) were evaluated. Plasma ACTH levels were not increased after the less intense exercise; on the contrary, ACTH concentrations increased significantly 10 min after the most intense exercise [Different studies have been focused on the responses of ACTH to different types of exercise, having demonstrated its reactivity to the most intense ones ,6. Furthexercise .As it has been reported, increases in circulating cortisol levels following exercise are related to the extent of stress experienced . ConsideOn the other hand, there is little information about the effects of vigorous exercise  on mental health and other psychological variables . The fewTaking into account all above mentioned, we performed this pilot study to address whether a definitive RCT was feasible with regard to (i) assess the impact of HIIT-based intervention on neurophysiological stress response, (ii) determine the subjects’ acceptability to specific HIIT, (iii) estimate possible changes on mood induced by HIIT intervention, and (iv) discover a possible connection between stress hormone response and mood variations after intense exercise.Thus, the primary objectives of this study were as follows:A. To assess how many subjects accepted the invitation to participate in this research and meet the inclusion criteria.B. To determine whether the eligibility criteria for participants were too open or too restrictive by estimating feasible eligibility and recruitment rate.C. To verify the impact of HIIT-based intervention on neurophysiological stress response and to assess the residual effects on main outcomes. D. To investigate the participants’ acceptability to a specific HIIT bout in terms of compliance to this intervention.E. To assess the acceptability of the outcome measures as methods to measure efficacy of the intervention within a definitive trial.The secondary objectives of this study were as follows:a. To evaluate the feasibility of blood sampling from participants’ antecubital vein in pre-, post-, and recovery period after HIIT intervention.b. To investigate the applicability of POMS questionnaire in pre-, post-, and recovery period after HIIT intervention.c. To measure precisely key outcome variables  and to calculate 95% confidence intervals for the difference between assessment time-points.d. To analyze the association between stress hormone responses and mood changes, from which the definitive RCT could be designed.Participants were students at the local Faculty of Educational Sciences, and they were recruited through announcements on social media and academic web sites. Interested participants then registered their interest with the researchers by telephone or email. This was followed up with a screening visit  with the researchers.Since this was a pilot study, a sample size calculation was not performed. The researchers aimed for more than 20 participants since previous studies reported high effect sizes for stress hormones and mood assessments using between 10 to 26 participants .The inclusion and exclusion criteria were as follow. Inclusion criteria: male with an age between 18 and 25 years; physically active ; good gNevertheless, applying inclusion and exclusion criteria we recognized that some participants were taking academic exams. This situation would lead them to experience anxiety or stress episodes, insomnia, and other symptoms which could interfere with the outcomes. We included this issue as exclusion criteria.2peak) with 1-min of passive recovery in-between. Finally, blood samples were drawn, and POMS questionnaire was filled by participants immediately and 30-min post-HIIT. Within 24 h before testing sessions, participants were instructed to avoid strenuous physical activity, and also to abstain from food (overnight fasting), caffeine, cacao, and alcohol 12 h before testing.Considering that this pilot study was conducted to gain experience in delivering the intervention, we decided to develop a quasi-experimental approach because this type of research design allowed us to evaluate the use and acceptability of the HIIT-based intervention and to assess biochemical and psychological outcomes despite limited resources that were available. Thus, a one-group pretest/posttest quasi-experimental design was used in this study in which each participant took part in two testing sessions separated by 5 to 7 days. Body composition analysis ; and a graded exercise test on a cycle ergometer (Ergoline Ergoselect 200) was performed during the first session. In the second session, after 15-min of sitting rest, venous blood samples were collected, and Profile of Mood States (POMS) questionnaire  was admiThis pilot study was approved by a local University Ethics Committee before the experiment was started (5 February 2017) and performed in accordance with the 1964 Declaration of Helsinki and its later amendments. Accordingly, all participants gave written consent after receiving information regarding the nature and purpose of the study.2 (VO2peak) on the cycle ergometer. After 5 min of cycling at 50 W, the workload was increased by 25 W per min until exhaustion (cadence: 70 rpm). Breath-by-breath pulmonary gas-exchange data were collected  and heart rate (HR) was continuously measured by telemetry . VO2peak was determined as the highest 20 s mean value reached before exhaustion, defined as the presence of two or more of the following criteria: (1) a maximal rating of perceived exertion (RPE): 19–20 points of 6–20 Borg’s scale [2 peak (pVO2peak) was registered for the HIIT intervention  a rrvention .HR and RPE monitoring. HR was continuously monitored during HIIT by a HR monitor . Mean (HRmean) and maximal HR (HRmax) were assessed for each cycling interval. Moreover, the Borg’s 6–20 scale [20 scale  was usedBiochemical analyses. 5-mL blood samples were collected 5 min before, immediately after, and 30-min post HIIT. Plasma ACTH levels were measured using ELISA techniques , with a detection range from 7.9–66.1 pg/mL . On the other hand, plasma cortisol concentrations were also analyzed by ELISA , with a detection range from 30–230 ng/mL, and cross-reactivity with prednisolone (5.1%), corticosterone (0.3%), and progesterone-estradiol (<0.1%). The inter and intra-assay CV were 8.6 % and 6.2%, respectively. All samples were analyzed in duplicate.Mood States assessment. To assess mood, the subjects completed the standard version of POMS [ of POMS  at pre-, of POMS :iPOMS = Nevertheless, according to the objectives of this pilot study, the following clarifications on measures must be considered: (a) the screening visit with the researchers was used to determine how many subjects accepted the invitation to participate in this research and meet the inclusion criteria; (b) percentage of subjects who meet the inclusion criteria was used to determine whether the eligibility criteria for participants were too open or too restrictive by estimating feasible eligibility and recruitment rate; (c) individual rates of perceived exertion were collected to check the impact of HIIT-based intervention on neurophysiological stress response; (d) the acceptability to a specific HIIT intervention was assessed by percentage of subjects who were not able to complete the entire HIIT bout; (e) the administration of a computerized version of POMS and duplicate analyses on ACTH and cortisol plasma levels were used to check the acceptability of the outcome measures as methods to measure effects of the intervention within a definitive trial.On the other hand, once this pilot study had commenced, we recognized that participants could be experiencing acute psychosocial stress  or blood sampling stress in one or both testing sessions. We therefore amended the protocol evaluating participants’ state anxiety levels at the beginning of the testing sessions   and alsor-value (r = Z/√N) [r < 0.1, small: r = 0.1–0.3, medium: r = 0.3–0.5, and large: r > 0.5. Bivariate correlations based on both individual raw data and delta percentages were also performed using the Spearman’s rho. For all tests, a p-value < 0.05 was considered statistically significant.Means and standard deviations (sd) were calculated for all variables. To check normality, Shapiro-Wilk test was conducted. Considering that data were not normally distributed, Friedman Test and Wilcoxon Signed Rank Test were used for intragroup comparisons . Moreover, effect size (ES) was calculated using  = Z/√N)  and was Recruitment began 19 March 2018 and closed on 9 April 2018. The final screening visit was on 4 May 2018. A total of 78 students were initially screened and 25 (32%) were finally included . The participants’ characteristics are summarized in 2peak was 45.3 ± 9.3 mL/kg/min and mean power output at this point (pVO2peak) was 275.0 ± 48.9 W. In session 2, all participants were able to complete the entire HIIT bout. Moreover, the state-anxiety scores (STAI) measured prior to HIIT were 17.50 ± 2.39, indicating low anxiety levels.max increased from 146.6 ± 11.6 bpm to 177.8 ± 10.9 bpm whereas HRmean increased from 126.3 ± 11.5 bpm to 159.5 ± 14.2 bpm . However, ACTH concentrations measured 30 min post-HIIT dropped significantly , but they were still higher than those observed in the pre-HIIT evaluation . As can be seen in p < 0.05, r = 0.716), reaching their highest levels 30 min post-HIIT .p = 0.036 and CI 95%: 0.333 to 2.787, p = 0.015, respectively). Unlike those dimensions, F-I and C-B increased significantly from pre-HIIT to post-HIIT  but they decreased from post-exercise to 30-min post-exercise . However, both A-H and V-A did not show significant changes throughout the three assessment time-points, although V-A scores measured at post-HIIT evaluation were the lowest. p = 0.002) showing a possible detrimental effect of intense exercise on mood. Although this effect could be explained by a slight decrease in V-A accompanied by significant increases in F-I and C-B observed in post-HIIT assessment time-point, the possibility of a harm effect of evident exercise-induced F-I increases on iPOMS scores must be taken into account for the future RCT study. This is especially significant when other POMS dimensions such as T-A, D-D, and A-H do not seem to be sensitive to high-intensity exercise.On the other hand, and as it can be also observed in p = 0.009), reaching pre-exercise values. Lastly, and considering the decreases in T-A, D-D, F-I, and C-B scores, iPOMS increased significantly after 30 min of recovery . In addition, ACTH and cortisol plasma levels were positively and significatively correlated with F-I  and negatively and significatively with iPOMS  in post-exercise situation. Moreover, ACTH showed a positive and significant relationship with C-B  at the same assessment time-point. Finally, a positive and significant correlation was observed between cortisol and C-B measured 30 min post-HIIT . On the other hand, using individual delta percentage of the difference between the assessment time-points, pre-post HIIT ACTH increases were inversely correlated with V-A decreases . Using raw individual data, ACTH and cortisol plasma levels were negatively and significatively associated with V-A in pre-exercise situation  and POMS dimensions such as fatigue and confusion after intense exercise. To our knowledge, there are few studies that have examined these relationships; nevertheless, our results seem to be consistent with some of them ,46, althLastly, there are some limitations in our study that should be mentioned. As it was previously indicated, the pretest/posttest quasi-experimental design used here has methodological weaknesses that do not allow properly addressing causality. Nevertheless, this pilot study was conducted according to the CONSORT 2010 Statement   to gain Moreover, stress associated with social evaluation threat or with blood sampling could affect significantly the outcomes of future RCT. To avoid these intervention biases it will be necessary (a) to evaluate the state-anxiety of subjects prior to each testing sessions, and (b) to apply distraction methods  during blood drawn in those participants who reported needle fear. Although in the present pilot study all reported state-anxiety levels were below the population median, it could be necessary to exclude from the analysis those participants who show high-levels of anxiety. Finally, measuring biases might be considered if iPOMS is used as mood states-related outcome. Although negative mood dimensions such as tension-anxiety, depression-dejection, anger-hostility, and confusion-bewilderment do not change after exercise, it would be possible that exercise-induced fatigue leads subjects to accentuate the fatigue-inertia dimension score, modulating in consequence the iPOMS score. Thus, if POMS is used, individualized analyses of each dimension are recommended. Additionally, the concurrent use of scales for estimating affective and enjoyment responses would allow us to obtain a more comprehensive understanding the potential role of high-intensity exercise in mood, affective feelings, and other related emotional states.Our data reflect the activities of only one pilot trial; in fact, the sample characteristics may have also limited the generality of our findings. According to the characteristics of our HIIT-based intervention, we only used physically active male college volunteers, so the results are not generalizable to females or sedentary populations. However, we hope that the methods may serve as a template for analyzing other pilot studies with similar or different designs in other exercise-related settings.According to the results obtained in this study, we can state that a single bout of HIIT increases the feelings of fatigue and confusion-bewilderment in healthy young males which could affect their mood states. This negative effect seems to be associated with HPA-axis activation and the increase of circulating levels of ACTH and cortisol. Future RCT studies should emphasize the influence of these stress hormones on HIIT mood responses by using specific ACTH or cortisol blockers and to evaluate the modulating effect of subjects’ exercise-related expectations, since in combination with the psychophysiological mechanisms involved in intense exercise, they could jointly determine the perceived and experienced psychological effects of exercise.


Breast cancer diagnosis at the initial stage of the disease considerably improves prognosis and survival rates. This retrospective study aimed to develop and validate a plasma microRNA signature as a non-invasive biomarker for early-stage breast cancer diagnosis. We confirmed in a testing cohort of 54 BC patients and 89 healthy volunteers the value of a signature based on miR-30b and miR-99a levels in plasma samples for stage I breast cancer detection. Furthermore, our results were blindly validated in a second cohort of 74 breast cancer and 74 healthy samples. The proposed microRNA signature presented high value as a fast, cost-effective, and non-invasive biomarker for early-stage breast cancer detection, which will lead to a better prognosis for breast cancer patients.The early diagnosis of breast cancer is essential to improve patients’ survival rate. In this context, microRNAs have been described as potential diagnostic biomarkers for breast cancer. Particularly, circulating microRNAs have a strong value as non-invasive biomarkers. Herein, we assessed the potential of a microRNA signature based on miR-30b-5p and miR-99a-5p levels in plasma as a diagnostic biomarker for breast cancer. This two-microRNA signature was constructed by Principal Component Analysis and its prognostic value was assessed in a discovery cohort and blindly validated in a second cohort from an independent institution. ROC curve analysis and biomarker performance parameter evaluation demonstrated that our proposed signature presents a high value as a non-invasive biomarker for very early detection of breast cancer. In addition, pathway enrichment analysis identified three of the well-known pathways involved in cancer as targets of the two microRNAs. Breast cancer (BC) is the most commonly diagnosed cancer in women and the leading cause of cancer-related death in most countries . The earCurrently, mammography is the standard breast screening procedure, but its efficacy for dense breasts and women under 40 years old is rather limited. In this context, other techniques such as ultrasounds, magnetic resonance imaging (MRI), or positron emission tomography (PET) may be used, but are not widely available as they are expensive techniques and can lead to over-diagnosis due to a lack of specificity in some cases . Thus, iIn the last few decades, microRNAs (miRNAs) have been proposed as important regulators of cellular activity. miRNAs are non-coding RNAs of 21–25 nucleotides that regulate gene expression at different levels and are involved in numerous biological processes. Focusing on cancer, miRNAs are deregulated in tumor tissues, where they may act as oncogenes or tumor suppressors by targeting genes involved in cancer-related processes such as tumor initiation, proliferation, cell death, angiogenesis, or invasion ,9,10,11.Several works described miR-30b-5p and miR-99a-5p to be potential non-invasive cancer biomarkers. Particularly, plasma circulating miR-30b-5p has been validated as a biomarker for the prognosis and detection of lung and breast cancer ,17,18,19In the present retrospective study, we sought to identify and to validate a two-miRNA signature based on the detection of miR-30b-5p and miR-99a-5p in plasma using Principal Component Analysis (PCA) to diagnose BC at a very early stage.This retrospective study enrolled plasma samples from healthy donors and BC patients from two different cohorts from independent institutions. The discovery cohort comprised 89 healthy donors and 54 BC patients from the Portuguese Oncology Institute of Porto , and the validation cohort included 74 healthy donors and 74 BC patients from Biomedical Research Institute INCLIVA and Valencian Biobanking Network (Spain). Plasma samples were collected in accordance with the Declaration of Helsinki. Peripheral blood was collected in EDTA-containing tubes and centrifuged at 2000 rpm for 10 min at 4 °C. Then, plasma was isolated and stored at −80 °C until further use. All BC plasma samples were collected before any treatment. The ethical committees of IPO-Porto (CES-IPOFG-120/015) and INCLIVA (2019/196) approved this study. Written informed consent was obtained from all participants.miRNA extraction from plasma samples was carried out using miRNeasy Serum/Plasma Kit  following the manufacturer’s protocol. RNA was quantified at the NanoDrop spectrophotometer  and stored at −80 °C until further use.A volume of 9.16 µL of RNA from plasma was retrotranscribed into cDNA using the High-Capacity cDNA Reverse Transcription kit , according to the manufacturer’s protocol. The reaction mixture was incubated at 16 °C for 30 min, at 42 °C for 30 min, and at 85 °C for 5 min in a thermal cycler. To determine miRNA expression levels, qRT-PCR was performed. A total of 2 µL of cDNA was amplified with 5 µL of Xpert Fast Probe 2× MasterMix , 0.5 µL of Taqman microRNA assays  and 2.5 µL of nuclease-free water. qRT-PCR reaction was carried out on a QuantStudio 5 Real-Time PCR System  under the following conditions: 98 °C for 3 min, 45 cycles of 95 °C for 10 s, 60 °C for 30 s, and 37 °C for 30 s. RNU38B  was employed to normalize the expression of miRNAs. A standard curve of four serial 10-fold dilutions of cDNA was run in each plate and used to calculate the expression of miRNAs. All samples were analyzed in triplicate.in silico analysis. This tool allows the identification of predicted miRNA targets as well as the significantly regulated KEGG pathways [To identify relevant targets of miR-30b-5p and miR-99a-5p, the online tool DIANA miRPath-v3.0 was used to perform an pathways . The algp-values < 0.05 were considered statistically significant. Differences in miRNA expression between two groups were evaluated by using the Mann–Whitney U non-parametric test. Kruskal–Wallis test was performed to evaluate associations between miRNA expression and clinical variables. The combined signature was calculated using Principal Component Analysis (PCA) in the discovery cohort and applied to the validation cohort. To evaluate whether miRNAs individually or the combined signature had prognostic potential, optimal cut-point was calculated using Youden Index . ReceiveHerein, we propose a combined signature of miR-30b-5p and miR-99a-5p levels in plasma as a candidate biomarker to diagnose very early-stage BC; very early stage was defined as TNM stage I . First, To assess miRNAs’ circulating levels in plasma, a set of 89 samples from healthy donors and 54 plasma samples from BC patients from IPO-Porto (discovery cohort) was selected. Clinicopathological data from the discovery cohort are detailed in p = 0.0023) (p < 0.0001) (p < 0.0001). Circulating miR-30b-5p levels were significantly higher in samples from BC  compared to healthy controls  ( 0.0023) A. In the 0.0001) B. Furthep < 0.0001) (p < 0.0001), and biomarker parameters of 57.4% sensitivity, 87.54% specificity, and 76.22% accuracy were obtained  than in plasma samples from healthy controls  ( 0.0001) A. In addobtained B.p < 0.0001). ROC analysis showed an AUC of 0.9273  and biomarker performance parameters of 82.35% sensitivity, 87.54% specificity, and 86.79% accuracy  than in healthy donors   (p = 0.0235)  than patients with more advanced disease  (= 0.005) A. Accord 0.0235) B. These n = 18) were selected. Clinicopathological data from the validation cohort are detailed in p = 0.0004)  A. ROC an 0.0004) B.To further explore the association of these circulating miRNAs and clinical behavior, functional enrichment analysis based on KEGG annotation allowed for the identification of the pathways simultaneously targeted by miR-30b-5p and miR-99a-5p. Three pathways surfaced as simultaneously regulated by these two miRNAs: p53 signaling pathway, Wnt signaling pathway, and pathways in cancer A. ImportThe detection of BC in the initial stages allows for increased long-term survival rates. In this context, mammography is the main approach for BC screening, regardless of its limitations . HoweverRecent studies proposed liquid biopsy as a valuable procedure for cancer detection that might complement current clinical tools, improving not only diagnosis but also treatment monitoring . The mosOn the other hand, circulating miRNAs and EVs are still in development as non-invasive biomarkers. miRNAs have high stability in body fluids and have been demonstrated to be deregulated in most cancer types even at initial stages. Due to these facts, the detection of circulating miRNAs has emerged as a promising liquid biopsy strategy. Thus, several studies have evaluated circulating miRNA expression levels in different types of cancer as prognostic, predictive, diagnostic, and disease monitoring biomarkers . Particularly, circulating miRNAs are of major interest in cancer detection as they have been proved to be released by malignant cells and to be differentially expressed in body fluids from healthy donors and cancer patients. Nowadays, circulating miRNAs in plasma have been demonstrated to have the potential to detect cancer even in the earliest stage of the disease . In thisHerein, we propose a two-miRNA signature based on the expression of miR-30b-5p and miR-99a in plasma samples as an early-stage BC detection biomarker. Our data show the potential value of this signature in distinguishing particularly stage I BC without any clinical manifestation from healthy samples. ROC analysis in the testing cohort of 106 individuals presented an AUC of 0.92 and 82.35% sensitivity, 87.54% specificity, and 86.79% accuracy. Moreover, we blindly validated our proposed signature in a second cohort of 92 individuals from an independent institution and our results demonstrated the value of the signature as a diagnostic biomarker with an AUC of 0.76, 77.77% sensitivity, 68.91% specificity, and 70.65% accuracy. Given the importance of an early diagnosis to improve prognosis and survival rates, we must highlight the higher performance achieved by our signature in stage I BC compared to all BC patients. In addition, this non-invasive biomarker could be detected by an efficient, fast, and non-expensive liquid biopsy method in the very initial stage of the disease when patients could still be asymptomatic.TP53, coding for the protein p53, is the most frequently mutated gene in cancer and it is linked to hereditary disease [TP53 is the second most frequently mutated gene after PI3KCA and it is decisive in early events of the development of BC and in the process of advanced disease. These characteristics make TP53 mutations a prognostic and predictive biomarker in BC and also a potential target for new treatments [To further elucidate the biological relevance of these circulating miRNAs, we also analyzed the pathways commonly targeted by both miR-30b and miR-99a. Interestingly, our in silico analysis identified three significantly targeted pathways: p53 signaling pathway, Wnt signaling pathway, and pathways in cancer, which have been widely implicated in breast carcinogenesis, as well as other types of cancer. The p53 pathway is one of the most important dysregulated pathways in cancer and it governs a complex anti-proliferative transcriptional program.  disease . p53 cas disease . The los disease . Particueatments ,58.The Wnt pathway is a key cascade in breast oncogenesis due to its implication in mammary gland development and morphogenesis. Wnt/β-catenin pathway mutations are rare in BC. However, hyperactivated signaling is commonly found and associated with worse prognosis, particularly in triple-negative BC. The epigenetic inactivation of Wnt pathway antagonists, such as WIF1, which is reduced in 60% of BC, is also frequent . Wnt casAltogether, our results obtained in the discovery cohort and validated in a second set of plasma samples support the value of this two-miRNA signature for detecting early-stage BC. Future evaluation of this signature in a bigger and multicenter study is warranted. The good performance of this panel makes it a good option to complement the standard BC screening techniques in a cost-effective manner. The evaluation of miRNA signatures in plasma would be a fast, reproducible, and effective test to improve the percentage of patients diagnosed with stage I BC, thus achieving better survival rates and prognosis for BC patients. Nonetheless, an important limitation of circulating miRNA signatures is the variability observed between detection methods such as RT-qPCR, microarrays, and deep sequencing and also between body fluids such as serum and plasma. Thus, the establishment of an accurate and reliable circulating miRNA signature for BC detection is still in development. These challenges indicate the need for the validation and standardization of miRNA assessment in liquid biopsy before the translation to clinical practice takes place. In the present study, the proposed two-miRNA-based signature was demonstrated to have high potential as a non-invasive biomarker for the early diagnosis of BC, essential for prognosis improvement. Although we were able to confirm the high diagnostic accuracy in two independent cohorts of BC patients, larger multi-institutional studies are needed to confirm its applicability in clinical practice.


A green technique was developed to extract hyaluronic acid (HA) from tuna vitreous humor (TVH) for its potential application in managing dry eye disease. Deep eutectic solvents (DES) were used to extract HA and were synthesized using natural compounds . The DES, the soluble fraction of TVH in DES (SF), and the precipitated extracts (PE) were evaluated for their potential use in dry eye disease treatment. In vitro experiments on human corneal epithelial cell lines and the effect on dry eye-associated microorganisms were performed. The influence of the samples on the HCE viability, their intracellular reactive oxygen species (ROS) scavenging capacity, inflammatory response, and antimicrobial properties were studied. According to the results, all samples displayed an antioxidant effect, which was significantly higher for PE in comparison to SF. Most of the tested samples did not induce an inflammatory response in cells, which confirmed the safety in ophthalmic formulations. In addition, the DES and SF proved to be efficient against the studied bacterial strains, while PE did not show an antimicrobial effect. Hence, both DES and SF at defined concentrations could be used as potential compounds in dry eye disease management. Nowadays, seeking ingredients from natural sources is on the rise as companies are more committed to the sustainable development of green products . The useThere are various techniques for efficiently extracting materials from biomass sources, including the use of novel deep eutectic solvents (DES) . These sStaphylococcus aureus and Pseudomonas aeruginosa, to assess whether the samples may also reduce bacterial growth, which would be helpful in the management of dry eye cases prone to infections.In this work, DES were prepared using lactic acid, fructose, and urea to extract hyaluronic acid (HA) from the marine raw material tuna vitreous humor (TVH). The use of DES replaces the time-consuming and/or conventional toxic methods to extract natural HA. Various techniques have been applied to extract HA from natural sources, such as terrestrial and marine biomass ,13,14. HProteus vulgaris (CAS: 9024-13-9), and sodium chloride  were obtained from Sigma-Aldrich .The following reagents were used in the extraction and chemical characterization processes. Ethanol absolute , hyaluronic acid disaccharide , DL-lactic acid , sodium phosphate , urea (CAS: 57-13-6), Chondroitinase ABC from 2DCF-DA) dye (CAS: 4091-99-0) was obtained from Merck Life Sciences . Tumor necrosis factor-α  and the IL-6 (REF 950.030.192) and IL-10 (REF 950.060.192) ELISA kits (Diaclone) were obtained from bioNOVA cientifica s. l. .The following reagents were used in cell-based studies. Penicillin-streptomycin, insulin (CAS: 11061-68-0), epidermal growth factor , benzalkonium chloride (8001-54-5), and phenazine methosulfate  were obtained from Sigma-Aldrich . Dulbecco’s Modified Eagle Medium (DMEM)/F-12 + GlutaMAX-I, fetal bovine serum, tetrazolium salt -2H-tetrazolium-5-carboxanilide, XTT, (CAS: 298-96-4), the BCA protein kit, and DMEM/F12 without phenol red were obtained from Thermo Fischer Scientific . Cell permeant 2′,7′-dichlorodihydrofluorescein diacetate , and Clorocil eye drop, with 8 mg/mL chloramphenicol .The following reagents were used in the microbial studies. Cation-adjusted Mueller Hinton broth (CAMHB) was obtained from BD , trypticase soy agar (TSA) and trypticase soy broth (TSB) were obtained from VWR . Gram-positive bacteria The raw material tuna eyes were kindly donated from Tunipex, Faro, Portugal. They were kept frozen and were cut to separate the tuna vitreous humor (TVH) from the eyeball. The frozen vitreous humor was then freeze-dried and stored at −20 °C until needed.LA:F) and lactic acid:urea (DESLA:U) at a molar ratio of (5:1) and (4:1), respectively. The freeze-dried raw materials were mixed with the prepared solvent systems at a mass ratio of 1:100 , and they were stirred for 24 h at 50 °C [LA:F and SFLA:U). Precipitation of the dissolved extracts was performed by the addition of 3 times the DES volume of ethanol as an anti-solvent [LA:F and PELA:U). The experiments were performed in triplicate.The DES were synthesized by combining the natural components at specific molar ratios under heating at 80 °C and stirring for 30 min until a homogeneous liquid system was obtained. The systems prepared were lactic acid:fructose . The long-chain HA was hydrolyzed using the enzyme chondroitinase ABC to obtain its disaccharide unit. The hydrolysis was performed by treating 40 μg of the sample with 25 mU of chondroitinase ABC in a sodium phosphate buffer  at 37 °C for 3 h . The enz2 atmosphere at 37 °C. The cellular viability was assessed using the XTT-based colorimetric assay. HCE cells were seeded in 96-well plates, starting with the 38th cell passage, and grown to 90% of pre-confluence. After 24 h, the cells were maintained in a non-supplemented and serum-free medium for another 24 h. A stock solution of each compound was prepared in pure cell culture medium and filtered using sterile 0.2 µm filters. The prepared stock solution concentration was 12 mg/mL for the DES samples and 4 mg/mL for the SF and PE samples. Serial dilutions of the tested compounds were performed in the plates. The cells with the treatments were kept for 24 h at 37 °C. Cells that were treated with the culture medium were used as the negative control, and cells treated with benzalkonium chloride (0.005% w/v dissolved in culture medium) were used as the positive control. To perform the XTT colorimetric assay, 100 µL of DMEM/F12 without phenol red was added to each well, followed by 25 µL of XTT and PMS mixture prepared prior to its use by adding 10 µL of PMS to the 1 mg/mL XTT solution. Incubation was performed at 37 °C for 3 h. The absorbance was then assessed using a UV/Vis microplate multi-reader at 450 nm and 620 nm . The experiments were performed in triplicate for each condition. The percentage of viable cells in the treated cells in comparison to the control was computed using the following equation:The two lactic acid-based DES and their corresponding SF and PE testing samples were evaluated in vitro on human corneal epithelial (HCE) cell line . The HCE2DCF-DA dye to measure intracellular reactive oxygen species (ROS) levels in UV-B radiation-exposed cells. This non-fluorescent dye diffuses in the cells and is cleaved into H2DCF that is further oxidized to fluorescent DCF by the ROS. The HCE cells were cultured in 24-well plates and grown to 90% of pre-confluence. They were then maintained in a medium that is serum- and supplement-free for 24 h at 37 °C. The cells were then pre-treated with the compounds for 1 h at 37 °C at specific concentrations selected based on the results obtained from The antioxidant effect of the compounds was analyzed using the HThe potential inflammatory effect of extracted compounds on corneal epithelial cells was evaluated in vitro, measuring cytokine/chemokine secretion after HCE cells stimulation using TNF-α. Similar to previous studies, HCE cells were cultured in 24-well plates and grown to 90% of pre-confluence, then maintained in a serum- and supplement-free medium for 24 h at 37 °C. The cells were then pre-treated for 2 h at 37 °C with the compounds at specific concentrations. Following this, TNF-α at a concentration of 25 ng/mL was added to the cells for 24 h. Cell supernatants were collected after 24 h, and interleukins IL-6 and IL-10 production quantified with human interleukins (IL-6 and IL-10) ELISA kits according to manufacturer’s instructions. Interleukins’ concentration in each well was normalized to total protein content determined by the BCA protein assay kit. The experiments were performed in triplicate, and the samples were performed in duplicate.p values < 0.05 were considered statistically significant. GraphPad Prism software  was used for the figure plotting.The statistical analysis of the biocompatibility studies on the HCE cell line was analyzed using the SPSS software package . Results were expressed as mean ± standard error of the mean (SEM). One-way analysis of variance (ANOVA) with Tukey’s post hoc test or Games-Howell test was used for intergroup comparisons. S. aureus and P. aeruginosa. The stock solutions of the DES, SF, and PE testing samples were prepared by dissolving the samples at specific concentrations . In addition, the minimum bactericidal concentration (MBC) was determined by plating 100 μL from the wells with no growth on a TSA medium and incubating for 16–24 h at 37 °C. The MBC can be defined as the lowest concentration that killed at least 99.9% of the bacteria over the period of the assay and is complementary to the MIC. Results of the MIC and MBC were expressed as the median value obtained for each of the three replicates. A test was performed using a two-fold serial dilution of the samples with the addition of sterile medium to ensure their sterility.The assay was performed based on the broth microdilution method according to the CLSI M07-A10 guidelines . In brieDES were used for the extraction of HA from TVH, a natural marine by-product that is abundantly discarded. This method could not only ensure the use of natural HA for a therapeutic purpose but also implement the valorization of the discarded TVH in a green and low-cost process using DES. HA is a highly valuable biopolymer that has shown increased use in pharmaceutical formulations, namely in tear formulations for dry eye treatment . For insLA:F and DESLA:U, respectively). LA:F and SFLA:U), and the extracts were then precipitated (PELA:F and PELA:U). All samples of the process  were tested to evaluate their bioactivity. This allowed the evaluation of DES, not only as an extracting solvent but also as part of a therapeutic system containing the soluble fraction of TVH in DES. The comparison between the SF and PE samples was performed to study the differential bioactivity between the compounds. This also allows assessing a possible combined effect of DES with the soluble extracts since enhanced bioactivity, in this case, can eliminate the requirement for extract precipitation from DES, which consequently decreases the process time and cost. The extracts characterization is shown in LA:F and PELA:U, respectively . In contrast, SFLA:U and PELA:U had the same highest viable concentration of 0.5 mg/mL.The effect of each testing sample on cell viability of HCE cells was studied at different concentrations, as shown in p < 0.001) in the testing samples, as shown in LA:F, the production of ROS in the highest tested concentration (1 mg/mL) significantly decreased in comparison to the control. No antioxidant effect was observed for the studied doses of DESLA:U. A significant decrease in ROS production was observed for SFLA:F and SFLA:U in comparison to the control at concentrations of 0.5 and 0.75 mg/mL for SFLA:F, and 0.25 and 0.5 mg/mL for SFLA:U. Furthermore, PELA:F and PELA:U showed antioxidant capacity at different tested concentrations, including low concentrations of 0.062 and 0.125 mg/mL for PELA:F and PELA:U, respectively. Hence, the precipitation of the bioactive ingredients extracted from TVH displays a significant antioxidant property.The antioxidant effect of the samples was evaluated to analyze their potential use in ophthalmic therapy. Previous studies have demonstrated that HA-containing eye drops could decrease oxidative stress and inflammation, which consequently improves dry eye symptoms . An antiLA:F sample in comparison to SFLA:F. Therefore, the PELA:F samples are the most promising compounds having a higher significant antioxidant effect.To compare the antioxidant effect between the testing samples, the percentage of detected ROS was quantified in comparison to the control . A higheLA:F at 0.5 mg/mL (Previous studies have demonstrated that low molecular weight HA has pro-inflammatory properties ,52. The .5 mg/mL . TherefoS. aureus and P. aeruginosa [S. aureus and P. aeruginosa are shown in Dry eye disease caused by tear deficiency or excessive tear evaporation conditions is often associated with ocular surface conditions, such as meibomian gland dysfunction, anterior blepharitis, keratitis, among others. These conditions lead to modifications in the ocular surface in the type and concentration of bacteria, including ruginosa . Hence, LA:F and PELA:U, respectively. These results suggest that DES testing samples are indeed the most promising compounds to be used for antimicrobial effect against the 2 selected bacterial strains, as their MIC and MBC values ranged from 4 to 16 mg/mL. Previous studies demonstrated that lactic acid, fructose, and urea were shown to have an antimicrobial effect. For instance, lactic acid was proven to be an effective compound for the treatment of both S. aureus and P. aeruginosa [S. aureus, and its administration in an adjunctive therapy treated P. aeruginosa infection [S. aureus growth [It was possible to observe that SF samples presented a similar range of MIC and MBC values in comparison to DES. Hence, the presence of the TVH soluble fraction in DES did not improve its antimicrobial effect remarkably. A decreased antimicrobial activity was detected for the PE testing samples towards both bacteria since MIC and MBC values were higher than those of the DES and SF samples. In that case, these values were above 500 µL for PE sample/mL, the highest concentration tested, corresponding to 148 and 76 µg HA/mL for PEruginosa . Similarnfection ,57. Ureas growth . Furthers growth . This wos growth ,47. Frucs growth ,42,59.Regarding the tested commercial eye drops, it was observed that the HA-based commercial eye drops did not show antimicrobial activity against the microorganisms studied. This result is expected since the function of this product is not anti-infective, as HA does not act as an antimicrobial agent and is mainly efficient to maintain ocular hydration and comfort ,60. On tLA:F sample in comparison to the SFLA:F sample, showing a significant decrease of the ROS in comparison to the control. All testing samples did not display a pro-, nor anti-inflammatory response, based on the studied IL-10 and IL-6 levels, except for SFLA:F testing sample at 0.5 mg/mL, which significantly increased IL-6 secretion. Furthermore, DES and SF samples showed antimicrobial activity against dry eye-associated bacteria S. aureus and P. aeruginosa. In conclusion, DES and SF samples seem to be the most promising samples, and they could be used in ophthalmic therapeutic applications due to their antioxidant and antimicrobial activities. Consequently, the precipitation of the extracts from DES would not be necessary in order to obtain bioactive ingredients, which could reduce processing costs and time for future applications.The current work presents a process that contributes to the valorization of inedible food waste. A green technique was applied to obtain a HA from TVH using lactic acid-based DES for the valorization of industrial by-products. The DES, SF, and PE testing samples were tested in vitro for their potential application in dry eye disease therapy. A range of concentrations of the samples was studied to analyze the antioxidant and anti-inflammatory response. A higher antioxidant effect was observed for the PE


Plasmodium knowlesi in a bacterial system. Synthetic P. knowlesi lactate dehydrogenase (Pk-LDH) gene was cloned into pET21a expression vector, transformed into Escherichia coli strain BL21 (DE3) expression system and then incubated for 18 h, 20 °C with the presence of 0.5 mM isopropyl β-d-thiogalactoside in Terrific broth supplemented with Magnesium sulfate, followed by protein purifications using Immobilized Metal Ion Affinity Chromatography and size exclusion chromatography (SEC). Enzymatic assay was conducted to determine the activity of the enzyme. SDS-PAGE analysis revealed that protein of 34 kDa size was present in the soluble fraction. In SEC, a single peak corresponding to the size of Pk-LDH protein was observed, indicating that the protein has been successfully purified. From MALDI-TOF analysis findings, a peptide score of 282 was established, which is significant for lactate dehydrogenase from P. knowlesi revealed via MASCOT analysis. Secondary structure analysis of CD spectra indicated 79.4% α helix and 1.37% β strand structure. Specific activity of recombinant Pk-LDH was found to be 475.6 U/mg, confirming the presence of active protein. Soluble Pk-LDH that is biologically active was produced, which can be used further in other malaria studies.Plasmodium lactate dehydrogenase (pLH) is one of the enzymes in glycolysis with potential target for chemotherapy. This study aimed to clone, overexpress and characterize soluble recombinant lactate dehydrogenase from  Plasmodium: Plasmodium falciparum, P. vivax, P. malariae and P. ovale. However, zoonotic malaria infections are increasingly being reported. This new type of infection is caused by P. knowlesi—a species previously known to infect primates. The first naturally human infection was reported in Kapit Division of Sarawak in 2004 . In their study on Pk-LDH, Singh et al. (2012) determined the specific activity; however, the Vmax and Km value was not reported  (103 deg cm2 dmol−1). The K2D3 web server  was utilized to estimate the ratio of the secondary structure elements in the CD spectra.The CD spectra of recombinant Pk-LDH, followed by purification of homogeneity and protein characterization. We have successfully obtained 0.2 mg of pure active recombinant Pk-LDH protein from 300 mL of bacterial culture. The MALDI-TOF analysis confirmed that the purified protein is similar to P. knowlesi lactate dehydrogenase even though the gene was obtained through a synthetic process. The recombinant Pk-LDH was found to be active, as revealed by enzyme-specific activity analysis. The production process used for obtaining pure and active recombinant Pk-LDH in this work has the potential to be used in enzyme inhibition assay for anti-malarial drug development.In this work, we have reported the methods used for cloning and overexpression of


Vibrio vulnificus FJ03-X2 strain with high pathogenicity and strong virulence were established in zebrafish (Danio rerio) fibroblast cell line (ZF4 cells) and zebrafish. Zebrafish tnfb (Zetnf-b) gene was cloned and expressed by Escherichia coli BL21 (DE3), and Zetnf-b polyclonal antibody was prepared. Pre-injection of Zetnf-b polyclonal antibody and AG-126 before infecting with V. vulnificus could increase the survival rate of zebrafish by 36.6 and 46.7%, respectively. Pre-injection of Zetnf-b polyclonal antibody could effectively decrease the mortality of zebrafish infected by V. vulnificus. Thus, TNF polyclonal antibody therapy could be considered as an effective strategy to control V. vulnificus in fish.Tumor necrosis factor (TNF) plays an important role in an inflammatory cytokine storm. Over-secretion of TNF by the host in response to infection aggravates the disease. TNF expression level is positively correlated with the mortality caused by some bacterial infections. Therefore, using TNF antibody may alleviate the inflammation to resist bacterial infections. The function of fish TNF-b antibody in bacterial infection is still unclear. In this study, infection models of  Danio rerio) (Larimichthys crocea)  (Epinephelus coioides) (E. coioides) (Oncorhynchus mykiss) (Oryzias latipes) (Mycobacterium marinum (Aeromonas hydrophila and Edwardasiella tarda) and viral  infections (Fish tumor necrosis factor (TNF) family members are cytokines with multiple effects, which play vital roles in cell growth, differentiation, proliferation and immunity, apoptosis, macrophage activity and autoimmune diseases . Fish poo rerio) , large y crocea) , Japanesivaceus)  and grouoioides) . In addioioides) , rainbow mykiss) , zebrafilatipes) . There a marinum . It playfections . Zebrafifections –17. HoweVibrio vulnificus is the main pathogen of important economic fish, such as European eel (Anguilla Anguilla), large yellow croaker and grouper. As one of the most serious aquatic pathogens, it can cause hemorrhagic sepsis and the death of aquatic animals. Also, it can infect people through wounds or foodborne pathogenic infections . The result presented that the polyclonal antibody could significantly improve the survival rate of the immunized group after being infected with V. alginolyticus  were developed in the form of TNFα antibodies to treat many diseases, including osteoporosis, psoriasis, arthritis and Crohn disease. The TNF superfamily is considered as an active target for human drug development  in immune-related tissues of zebrafish in response to the intraperitoneal infection of V. vulnificus and that in the infected ZF4 cells at different time points. V. vulnificus infection models of ZF4 cells and zebrafish were developed to study the role of Zetnf-b during infection. Finally, the Zetnf-b gene was cloned, and the effect of its polyclonal antibody on resistance to V. vulnificus infection was determined in zebrafish. The results provide a perspective of TNF antibody therapy for the prevention and treatment of V. vulnificus in fish.This study aimed to investigate the function of zebrafish TNF-b polyclonal antibody in fighting against All zebrafish were anesthetized with tricaine methane sulfonate , and surgeries were performed. All animal experiments were conducted in accordance with the guidelines and regulations of the Institutional Animal Care and Use Ethics Committee of Fujian Academy of Agricultural Sciences (Permit Number: BI-AEC-2021055006).2 cell incubator. Sprague Dawley (SD) rats of Specific-pathogen-free (SPF) grade were purchased from the Laboratory Animal Center of Fujian Academy of Traditional Chinese Medicine, Fuzhou, China. The fish bacterial pathogen, V. vulnificus FJ03-X2 strain, which possesses strong virulence and high pathogenicity, was isolated from the sick European eel (Anguilla anguilla) and maintained in our laboratory. Its half-lethal dose (LD50) for elvers (15–30 g/tail) at a water temperature of 28°C was 2.3–7.1 × 103 colony forming unit (CFU)/tail. Tryptic soy broth (TSB) medium  was used to cultivate V. vulnificus FJ03-X2 strain.The zebrafish (AB strain) and ZF4 (ATCC CRL-2050) were purchased from the China Zebrafish Resource Center . The zebrafish with an average weight of 0.2 g were raised in a flow through pure water aquaculture system. No disease was observed during breeding. The ZF4 cells were maintained at 28°C in DME/F12 medium  with 10% fetal bovine serum  containing 10 U/ml penicillin and 10 μg/ml streptomycin in a 5% COZetnf-b-F and Zetnf-b-R primers (tnf-b gene (Accession no.: NM_001024447). Total RNA from liver and kidney of 3 healthy zebrafish was extracted using Trizol Reagent solution  according to the manufacturer's protocol. Zetnf-b was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into pMD18-T vector . The positive clone plasmid was subjected to double enzyme digestion and sub-cloned into the expression vector pET-32a  to get recombinant expression plasmid pET32a–Zetnfb. The recombinant proteins of Zetnfb (rZetnfb) were obtained by Escherichia coli BL21 (DE3) containing pET32a–Zetnfb and confirmed by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). primers  containiThe rZetnfb was purified by cutting the target band from the SDS-PAGE gel and quantified. T 10 basic ULTRA-TURRAX ®  was used to mix the target protein and adjuvant in a volume ratio of 1:1. Complete Freund's adjuvant  was mixed with rZetnfb for the first immunization, and incomplete Freund's adjuvant  was mixed with rZetnfb for the last immunization. Five SD rats of SPF grade were inoculated with the mixed substance through multiple subcutaneous injections to prepare polyclonal antibody. A part of SD rats collected before immunization was used as a negative control in the experiments. All the serum samples of SD rats were collected after the immunization. The titer of the serum polyclonal antibody was determined by enzyme-linked immunosorbent assay (ELISA) according to the method described previously . The spe2 and 95% air at 28°C. V. vulnificus FJ03-X2 cells in the logarithmic growth phase were diluted with (TSB) medium at a density of 1.0 × 106 CFU/ml. One group of cells without V. vulnificus inoculation was used as a negative control. The other three groups were inoculated with V. vulnificus for 12 h at three multiplicity of infection (MOI) of 1:1, 1:10 and 1:100, respectively. Optical density (OD) was measured at 490 nm (OD490) with a full-wavelength microplate reader . The percent cytotoxicity in each plate was calculated through the level of lactate dehydrogenase (LDH) using the standard procedure from CytoTox96 non-radioactive cytotoxicity assay . A low control (LC) and a high control (HC) were set up, and the percentage of cytotoxicity was calculated according to following equation:The ZF4 cells were plated in 6-well culture plates and divided into 4 groups with 3 wells per group. These were incubated overnight in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DME/F-12) in an incubator with 5% CO5 CFU/g, 5.61 × 104 CFU/g and 1.4 × 104 CFU/g V. vulnificus, respectively. The negative control group was injected with 10 μl of sterilized phosphate-buffered saline (PBS). The zebrafish status and survival rate were observed and recorded once every 3 h until 24 h. The LD50 of V. vulnificus infection in zebrafish was determined according to the recorded survival rate and the concentration of bacteria for challenging zebrafish.A total of forty zebrafish of similar size (Tail weigh of 0.22 g ± 0.02 g) and good growth condition were divided into 4 groups. According to the weight of the zebrafish, three groups were injected intraperitoneally with 1.4 × 10V. vulnificus [LD50 =4.39 × 104 (CFU/g) per fish]. The control group was injected with 10 μl of sterilized PBS. Kidney, muscle and intestine samples were collected from three fish of each group at different time points  for RT-PCR detection. Kidney samples from healthy and infected zebrafish at different time points  were collected for western blotting. The activity and survival rate were recorded.The experimental group was injected intraperitoneally with 10 μl V. vulnificus at different time points . Intestinal, muscle and kidney samples were collected from control and experimental groups. The TNF inhibitor AG-126 was purchased from Sigma-Aldrich . RNA extraction was conducted by using Trizol Reagent  according to the manufacturer's protocol. qRT-PCR was performed using TB GreenTM Premix Ex TaqTM II reagent Kit  with the Step Two TM Real-Time PCR System . Primers specific for Zetnf-b and β-actin are shown in Zetnf-b was calculated using the relative quantification analysis module of the 2Ct−ΔΔ method , and the protein concentration was detected by BCA protein content detection kit . Samples with equal amounts of protein were subjected to SDS-PAGE and transferred to polyvinylidine difluoride membrane . After blocking, the membrane was incubated with the primary polyclonal antibody of Zetnf-b , which was prepared as descripted in 2.4 and rabbit polyclonal anti-β-actin  at 4°C for 12 h. After washing, the membrane was incubated with goat-anti-mouse  and Goat Anti-mouse Rabbit  secondary antibodies for 2 h at 28°C. Detection was performed using an ECL hemiluminescence kit . After the film was scanned, the gray value of each protein band was measured, and data were presented as a ratio of the value to that for-actin. Zetnf-b expression in the kidney of mock-infected zebrafish was set to 1.0 and marked as 0 h, and the expression of Zetnf-b in infected zebrafish at 2–24 hpi was compared to the expression at 0 h.t test analysis. The marker * represents P < 0.05, which is considered to be statistically significant, and ** represents P < 0.01, which is considered to be significantly different.Experiments were performed in triplicate and the results were expressed as mean ± standard error (SE). All data were analyzed using Statistical Package for the Social Sciences (SPSS) for Windows (Version 16.0). The difference between the control group and the experimental group was tested by independent sample V. vulnificus infection were elongated, and these were in a healthy cell growth state with high fusion and adherence. Cells in the first group (MOI = 0.01) shrank significantly. Most of the cells in the third group (MOI = 1) lost their adhesion, and the cell structure was broken  was cloned  and extracellular protease (ECPase), can cause cell injury  was significantly up-regulated after stimulating by V. pelagius , IL-6, IL-8, and IL-10 . Sepsis nfection . In vivopelagius . In zebrdeclined . Moreoveuillarum . Our stuV. vulnificus infection, Zetnf-b polyclonal antibody or TNF inhibitor AG-126 was pre-injected to zebrafish. We found that pre-injection with Zetnf-b polyclonal antibody significantly increased the survival rate of V. vulnificus infected zebrafish. V. vulnificus infection in zebrafish. In addition, it confirmed that pre-injection of Zetnf-b polyclonal antibody aimed to inhibit TNF release or TNF activity, thereby reducing the inflammatory cytokine storm caused by bacterial infection and decreasing fish mortality, which is an effective attempt to treat bacterial infection in zebrafish.However, the overwhelming production of inflammatory cytokines can lead to organ dysfunction and eventually death. To confirm the function of cytokine Zetnf-b in fighting against Zetnf-b plays an important role in inflammatory storms induced by V. vulnificus infection. Pre-treatment of Zetnf-b polyclonal antibody and TNF inhibitor AG126 can help zebrafish to resist bacterial infections and improve the survival of zebrafish. This study provides a useful reference for the study of the biological characteristics of TNF, especially TNF-b, in important aquaculture fish and presents the TNF antibody therapy for the prevention and treatment of the V. vulnificus in the aquaculture industry.In conclusion, our study demonstrates that NM_001024447.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number can be found below: accession no.: V. vulnificus infection model in ZF4 cells and zebrafish, and also prepared the TNFb polyclonal antibody. HC was responsible for ZF4 cell and V. vulnificus culture. LZ and LK performed the experiment of pre-injecting the TNFb polyclonal antibody in zebrafish before V. vulnificus infection. CL reviewed the manuscript and provided critical suggestions. All authors discussed the results and approved the final manuscript for publication.XC and CL were responsible for the conception of the study. SL performed the cloning, expression, protein purification of zebrafish TNFb, performed data interpretation and wrote the manuscript. CJ performed the This work was funded by the Fujian public welfare research project (2019R1027-5). Meanwhile, this work was supported by the National Nature Science Foundation of China , Innovation Team Projects of Fujian Academy of Agricultural Science (STIT2017-03), Special Fund for Public-interest Scientific Institutions of Science and Technology Plan Projects of Fujian Province (2018R1019-7), and Provincial special support for high-level talents Double Hundred Plan special project.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The resulted doped Carbon Dots should preserve the anti-tumoral properties while gaining magnetic resonance imaging properties. Methods: Normal and cancer cell lines have been treated with doped Carbon Dots, and the cell viability has been measured. The doped Carbon Dots that exhibited the most prominent anti-tumoral effect accompanied by the lowest toxicity have been further in vivo tested. Magnetic resonance imaging evaluates both in vitro and in vivo the possibility of using doped Carbon Dots as a contrast agent. Results: According to the results obtained from both the in vitro and in vivo experimental models used in our study, Mn2+-doped Carbon Dots (Mn-CDs-NHF) exhibit anti-tumoral properties, do not significantly impair the cell viability of normal cells, and reduce lung metastasis and the volume of mammary primary tumors while allowing magnetic resonance imaging. Conclusions: Our findings prove that Mn-CDs-NHF can be used as theranostics agents in pre-clinical models.Background: Theranostics, a novel concept in medicine, is based on the use of an agent for simultaneous diagnosis and treatment. Nanomaterials provide promising novel approaches to theranostics. Carbon Dots have been shown to exhibit anti-tumoral properties in various cancer models. The aim of the present study is to develop gadolinium, Fe Due to the fact that cancer still remains a major worldwide health problem despite the latest advances in understanding cancer biology and developing new tools for early detection and treatments, researchers are looking for new ways to improve drug delivery and treatments toward being less invasive as well as having fewer side effects and higher efficiency .Different technologies, such as nanomedicine, have opened up new opportunities for the treatments in different diseases. Nanocomposites play an important role in drug release and biodistribution in various types of diseases. For example, they could improve the release of chemotherapeutic drugs in the therapy of certain cancer types . NanoparIn this context, Carbon Dots-based materials have gained attention in the scientific community due to their physical and chemical properties, which make them one of the most promising tools for drug delivery and imaging ,9. Cytot3+). However, due to the fact that Gd3+ is associated with different side effects such as nephrogenic system fibrosis, new alternatives are necessary [Despite the outstanding advances in medical imaging for diagnosis, magnetic resonance imaging (MRI) remains one of the most valuable techniques for in vivo investigations, due to its non-invasive nature in the absence of ionizing radiation, which may provide both anatomical and functional data . The majecessary ,19.3+ [3+ [Manganese-based nanoparticles (Mn-based NPs) represent promising alternatives due to their lower toxicity compared with Gd3+ ,21. Addi3+ [3+ ,23.Apoptosis or programed cell death is a regulatory system having a crucial role in controlling cell proliferation, tissue homeostasis, and removing cells that are no longer needed. The dysregulation of apoptosis mechanisms contributes to human diseases such as cancer. Molecules responsible for apoptosis have offered new ways to develop solutions in order to increase cancer cell death. Mitochondria play an important role in apoptotic cell death. Mitochondria dysfunction leads to apoptosis ,25.The cytokine interleukin-6 (IL-6) plays a crucial role in both normal human physiology and diseases. The findings demonstrate that IL-6 promotes the development of breast , colorec3+, Mn2+, or Fe3+. Using an MRI in vitro model, we have investigated the anti-tumoral effects of doped CDs-NHF as well as the imaging properties. The most promising candidate, Mn-CDs-NHF, has been further investigated in an in vivo 4T1 murine breast cancer model in order to evaluate its theranostics potential. Additionally, we have also aimed to explore the effect of the Mn-CDs-NHF on lung metastases due to the fact that lungs represent the primary metastasis site of the 4T1 cell line.In the present study, we aim to develop a theranostics agent that combines the anti-tumoral properties of CDs-NHF with enhanced imaging properties, which are doubled by low toxicity of Mn. For this, we have developed doped CDs-NHF with Gd2 were sourced from Merck Chemicals. High-purity water and reagent grade ethanol (EtOH) were used for all preparation stages. Briefly, the first stage involves the preparation of a Mn(II)–NHF complex at a ½ metal/ligand ratio, according to the following reaction:The preparation of the doped CDs-NHF was performed according to the procedure and the experimental setup mentioned in our previous work . N-HydroThe complexation reaction undergoes at 35–40 °C, under stirring for about 24 h in a water/EtOH (60/40% volume ratio) medium. The precipitate is collected, washed twice with water, and further freeze dried. In the second stage, the prepared complex is thermally processed through pyrolysis in the same conditions and process parameters using the previously mentioned experimental setup . The resThe mean zeta potential of the samples was evaluated by DelsaNano C Submicron Particle Size Analyzer from Beckman Coulter Inc. Light source: Dual 30 mW laser diodes as 658 nm. For zeta potential, the instrument uses electrophoretic light scattering (ELS) to measure the zeta potential, which determines the electrophoretic movement of charged particles under an applied electric field.®, St. Louis, MO, USA). Normal human mammary epithelial (HMLE) cells (ATCC), a generous gift from James Lorens , were cultured in DMEM/F12 supplemented with 5% horse serum, 20 ng/mL EGF, 10 ug/mL insulin, 0.5 ng/mL hydrocortisone, 10 ng/mL cholera toxin, and 1% Pen/Strep. Cells were seeded into a 96-well flat-bottom tissue culture plate at a density of 3000 cells/well and allowed to adhere to the plate by incubating at 37 °C under 5% CO2 overnight. Following overnight cell attachment, the cells were incubated with the tested doped CDs-NHF at 5% concentrations (50 µg CDs-NHF/mL) for 72 h. For each doped CDs-NHF, we have tested the amount of compound of that contained 50 µg CDs-NHF/mL.Murine breast cancer 4T1cells , a generous gift from James Lorens , were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (Sigma-Aldrich® Cell Viability Assay (Promega). After each of the 72 h treatment time periods, 50 μL of cell viability solution was added to each well, and the plate was reincubated for 4 h before luminescence recording using a multiplate microplate reader .For cell viability assessment, we used the CellTiter-BlueThe experiments were approved by Ethical Committee of the “Grigore T. Popa” University of Medicine and Pharmacy of Iasi and were performed in accordance with the European legislation on the protection of animals used for scientific purpose and with authorization from the National Sanitary Veterinary and Food Safety Authority (no. 17/09.04.2020). Female BALB/c mice  were used. The mice were housed in the animal facility of the CEMEX, “Grigore T. Popa” University of Medicine and Pharmacy, Iasi; in individually ventilated cages (IVCs) in a climate-controlled: 20 ± 4° Celsius, 50 ± 5% relative humidity, and 12 h light/dark cycles containing shaving bedding material with regular rodent chow and water ad libitum.6 in 50 μL) and injected into the mammary pad of female BALB/c mice under depth anesthesia, as previously described [n = 6) or Gadovist (n = 6) for 3 weeks. CD-NHF concentration is relative to mice blood volume and represents one of the previously tested concentrations [4T1 mouse breast carcinoma cells were suspended in RPMI medium/Matrigel (1:1) , and primary tumors and various organs  were collected. Half of each primary tumor and half of each lung were immediately suspended in RNA Save  and stored at −80 °C until use. The other half from each primary tumor and each lung were preserved in 10% paraformaldehyde (Sigma-Aldrich) for further analysis.The in vitro experiments were planned to evaluate the potential of the new prepared doped CDs-NHF as MRI contrast agents by determining the values of longitudinal and transversal relaxivities, r1 and r2, respectively, which were calculated from the corresponding T1 and T2 relaxation times. From each compound, a 5 mg/mL stock solution was prepared by dissolving 20 mg of powder in 4 mL of PBS 0.01 M, at physiological pH (7.4). The samples for MRI scanning were prepared by dispersing 10 ÷ 60 µL stock solutions in 10 mL of 1% agarose gel freshly prepared, at about 50 °C to obtain six concentrations for each tested compound, as follows: 0.005, 0.01, 0.015, 0.02, 0.025, and 0.03 mg/mL.The T1 and T2 measurements were performed using a 1 Tesla instrument for small animals , having B0 magnetic field shimming and coil calibration at a water proton frequency. The glass vials comprising the samples to be analyzed were positioned horizontally on the rat body acquisition bed in the center of the coil field of view. T1 relaxation times were determined by a two-point estimation method  through Two groups of tumor-bearing mice , comprising 6 mice per group, were investigated once per week by MRI scanning: one group named “Treated”, which has received Mn-CDs-NHF as a teranostic agent via i.p. administration and another group named “Control”, receiving only Gadovist, which is a commercial contrast agent. Both Mn-CDs-NHF and Gadovist were administered by i.p. injection 2 h prior to the MRI examination.®, Budapest, Hungary), and maintenance anesthesia was adjusted to 2%. The animals were subjected to MRI scanning using T1GRE sequence, coronal, and axial planes. This sequence was selected due to the short investigation time: 22 min for both planes. The parameters were as follows: TR = 13.1; TE = 3.8/2.2; FA = 30°; NSA = 4; slice thickness = 1 mm. The raw imaging data were processed using Carimas software (v. 2.10), which allowed the calculation including the tumor volume, apart from 2D images for each section.Fasted mice (not more than 6 h) were anesthetized using an isoflurane delivery system connected to a transparent chamber at a concentration of 3.5–4% isoflurane in a mixture of air and oxygen. After induction, the mice were transferred to the scanner bed of the nanoScan PET/MRI .For in vitro investigations, total RNA was isolated from the control and treated samples  using TRIzol reagent  according to the manufacturer’s protocol. Briefly, approximately 10For in vivo investigations, small fragments (≈1 mm/1.5 mm) of tissue were cut/scraped and diced with a sterile scalpel and homogenized prior to the RNA extraction. Briefly, tissue was homogenized in 1 mL of Trizol (Thermo Fisher Scientific) before the RNA was separated from DNA and proteins by adding 200 μL chloroform. Then, the total RNA was precipitated using 500 μL of isopropanol. The RNA pellet was washed afterwards with 1 mL of 75% ethanol solution and finally eluted in 11.5 μL ultrapure sterile water .A list of the primers used in this study, as well as the species, gene symbol, gene name, UniGene ID, and primer sequences is in IF was performed on 4 µm thick sections of formalin-fixed and paraffin-embedded tissues. Sections were deparaffinized and rehydrated; then, they were stained for detecting mitochondria in the fixed cells  or the apoptotic cells  according to the manufacturer’s recommendations.t-test for other statistical analysis. Quantitative data for statistical analysis were expressed as mean ± SEM (shown as error bar). Significance was established for p < 0.05.GraphPad Prism 6  software was used for statistical analysis. Grouped analyses were performed using one-way ANOVA for cell viability investigations and paired Student’s 2+-doped CDs-NHF (Mn-CDs-NHF) structure, which consists mostly of a carbonaceous core with a surface decorated with different remnant functional groups. The thermal destruction of the Mn(II)-NHF complex through the controlled pyrolytic process resulted in the formation of this graphitic-like core with a variety of functional groups attached on its surface, as previously studied [The structural analysis showed a typical Mn studied ,41. XPS Mn-CDs-NHF dispersed in water exhibit the same agglomeration tendency as observed in the case of non-doped CDs-NHF presented in our previous work , which wThe surface charge was determined by measuring the zeta potential of the synthetized CDs. The zeta potential value is determined by the stability of particles. Particles with a high zeta potential (either negative or positive) are electrostatically stable, while the particles with low zeta potentials tend to aggregate within a short period of time. The positive values of the zeta potential reflect the presence of a positively charged environment around the CDs. Having in mind the previously presented facts, the analyzed samples, CDs-NHF and Mn-CDs-NHF, which have positive values of zeta potential, confirm that both un-doped and doped CDs have positively charged surroundings. Moreover, it is obvious that the CDs-NHF sample possesses higher colloidal stability, having a mean zeta potential value of +16.59 mV, whereas the Mn-CDs-NHF sample possesses lower colloidal stability. The mean zeta potential value in this case is +6.87 mV, meaning that this sample tends to aggregate. The zeta potential measurement for each sample was acquired in triplicate, and the obtained values  are plotOur previous results  demonstr3+)-doped CDs-NHF (Gd-CDs-NHF) and Fe3+-doped CDs-NHF (Fe-CDs-NHF) significantly reduced the cell viability in both tested normal (HMLE) (The cell viability data demonstrate that gadolinium (Gdl (HMLE) A and canl (HMLE) B. Mn-CDsIn order to develop theranostics-doped CDs-NHF, in addition to their anti-tumoral properties, the imaging capabilities must be investigated. In our work, we have tested doped CDs-NHF as contrast agents in MRI .−1·mL·s−1, while r1 is insignificant to provide MRI contrast, of only 0.24 mg−1·mL·s−1. Since gadolinium is the only ion with clinical applicability to enhance the MRI contrast, it was considered in this study as a positive control, which was bound into the Gd-CDs-NHF in a similar manner to the rest of metal included in the prepared compounds. In such an environment, the Gd3+ ion caused relaxivity values of r1 and r2 of 9.96 and 22.13 mg−1·mL·s−1, respectively, suggesting a good ability to enhance the MRI contrast, especially in T2 sequences. Quite different is the case of Mn-CDs-NHF product, which although it has a relaxivity r2 significantly higher than all other materials tested, of 88.60 mg−1·mL·s−1, it also presents a noticeable value for r1, of 17.75 mg−1·mL·s−1, which is close to the r2 value of Gd-CDs-NHF. In such circumstances, we can infer that the Mn-CDs-NHF product is able to be successfully applied as a contrast agent in both T1 and T2 sequences, depending on what is pursued in that study. The in vitro imaging data using T1 and T2 MRI sequences are presented in The r1 and r2 relaxivity values represent the slope of the linear regression of T1 and T2 relaxation rates  in the range of the product concentrations we have considered. The relaxivity values indicate the T1/T2 shortening ability of the tested compounds with a direct impact on the MRI contrast. According to the r1/r2 values obtained, as it can be observed from Due to the fact that Mn-CDs-NHF exhibited the lowest impact on normal cell viability and the most effective impact on cancer cell viability, doubled by the fact that it can be successfully used as MRI contrast agent, we further investigate the Mn-CDs-NHF theranostics applications.Prior to accomplishing the MRI scans on breast cancer animal models, the effect of the selected compound (Mn-CDs-NHF) as a contrast agent on healthy mice was studied by comparing the resulting images with those obtained using Gadovist MRI images . The sam2+-based compound, which is predominantly in the liver, kidneys, peritoneal cavity, and heart and much less in the brain or other regions located distant from the injection site. The images are displayed in rainbow color visualization to show as suggestive as possible the difference in MRI contrast between different tissues, while the color intensities in all the recorded images were normalized using the same brightness level in all images so that they could be compared.An MRI signal is much more intense in the case of the group that received the suspension of Mn-CDs-NHF in comparison with the group receiving Gadovist. There is also a very obvious uptake of the MnRepresentative weekly MRI images of tumor-bearing mice are presented in In addition to the improved MRI contrast capabilities of Mn-CDs-NHF relatively to the commercial agent Gadovist, it is also essential to notice that when compared with the control group , a pro-inflammatory cytokine, mediates pro-survival signals in cancer and inflammations. Our in vitro investigation shows that the levels of IL-6 mRNA in cancer cell treated with Mn-CDs-NHF are significantly reduced relative to Gadovist-treated cells A.However, we failed to observe these changes in lung specimens B, which 3+, Fe3+, or Mn2+.The increase in the morbidity and mortality of cancer cases put pressure on the scientists to find new tools for diagnostic and treatments. The detection of cancer at early stages can have a massive impact on cancer treatment and patient status. Developing agents with theranostics properties, both therapy and imaging, will improve early cancer detection and overall cancer management. The importance of theranostic agents in the diagnosis and therapy of cancer has been recently reviewed ,44,45. SIn a recent investigation , 24 h inIn another recent study , Carbon 3+ or Fe3+ may reduce the anti-tumoral properties of CDs-NHF. The effect of Gd-CDs-NHF and Fe-CDs-NHF on both cell lines indicates that these two doped CDs-NHF exert a certain degree of toxicity. In addition to the in vitro data, the number of apoptotic cells is increased in the lung metastases in the Mn-CDs-NHF-treated group when compared with the classical MRI contrast agent, Gadovist. Even more, the decreased expression of the mitochondria staining in the Mn-CDs-NHF-treated group further sustains the anti-tumoral properties of Mn2+-doped CDs-NHF.Our in vitro findings showed that Mn-CDs-NHF exert a significant pro-apoptotic effect on cancer cell line without exhibiting a substantial impact on the normal cell line that we have tested. The other two doped CDs-NHF we have tested, Gd-CDs-NHF and Fe-CDs-NHF, significantly decreased the cell viability of the normal cell line. Moreover, the impact of Gd-CDs-NHF and Fe-CDs-NHF on cancer cell viability was lower relative to Mn-CDs-NHF, which indicates that doping CDs-NHF with Gd2+ preserves the anti-tumoral properties of CDs-NHF, we have investigated next the MRI imaging application, which is the second characteristic of a theranostics agent. Both the in vitro cell line investigations and in vivo data from healthy mice demonstrate that the MRI signals of Mn2+ from Mn-CDs-NHF fulfill the second condition required for Mn-CDs-NHF to be used as a theranostics agent in experimental models. In tumor-bearing animal investigations, the lung metastases from the control group are well developed, and MRI imaging of the Mn-CDs-NHF group demonstrates that treatment with Mn2+-doped CDs-NHF reduces the primary tumor volume. These data are in line with our newly reported in vitro pro-apoptotic effect of Mn2+-doped CDs-NHF and with our previous reported data that showed that non-doped CDs-NHF treatment reduces the Ki67cell proliferation marker at the level of lung metastases [As we demonstrated that doping CDs-NHF with Mntastases . The redThe MRI signal intensity of the liver, kidney and lung is enhanced in the Mn-CDs-NHF treated group relatively to healthy untreated mice, while the signal intensity of the brain and heart are at similar levels . In week3+-based compounds (Gd-CDs-NHF and Gadovist), Mn-CDs-NHF exhibited lower toxic effects on normal cells, anti-tumoral properties, and a stronger MRI signal.Altogether, our experimental findings demonstrate the theranostics properties of Mn-CDs-NHF. In comparison to Gd


A median load reduction of 23% was observed between 0–2 days and 3–5 days, and of 11% between 3–5 days and 6–9 days for the delay from onset of symptoms to saliva sampling. No significant median difference between no-symptoms vs. symptoms patients was observed. Charge was consistently similar for the majority of the clinical symptoms excepted for headache with a median load value of 3.78 log10 copies/mL [1.95–4.58] (P < 0.003). SARS-CoV-2 RNA viral load was associated with headache and gastro-intestinal symptoms. The study found no statistically significant difference in viral loads between age groups, sex, or presence de co-morbidity. Our data suggest that oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.The fast spread of COVID-19 is related to the highly infectious nature of SARS-CoV-2. The disease is suggested to be transmitted through saliva droplets and nasal discharge. The saliva quantification of SARS-CoV-2 in real-time PCR from asymptomatic or mild COVID-19 adults has not been fully documented. This study analyzed the relationship between salivary viral load on demographics and clinical characteristics including symptoms, co-morbidities in 160 adults diagnosed as COVID-19 positive patients recruited between September and December 2020 in four French centers. Median initial viral load was 4.12 log The fast spread of COVID-19 is related to the highly infectious nature of SARS-CoV-2. The disease is suggested to be transmitted through saliva droplets and nasal discharge . Indeed,Most published RT-qPCR assays for the diagnosis of SARS-CoV-2 are qualitative . ConsideClinicalTrials.gov (NCT04352959). One hundred and sixty subjects diagnosed as COVID-19 positive patient were recruited between September and December 2020 in four French hospital centers. The study protocol was reviewed and approved by the National Ethics Committee “Committee for the Protection of Persons South Mediterranean III,” France (2020.04.11 sept _20.04.06.46640). Written informed consent was obtained from all enrolled individuals. The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization–Good Clinical Practice guidelines.This analysis was a part of the randomized controlled trial BBCovid protocol published  and regiThe inclusion criteria were: (i) age 18–85 years-old, (ii) RT-PCR positive COVID-19 nasopharyngeal swab, (iii) asymptomatic or mild clinical symptoms for less than 8 days.The exclusion criteria were: (i) pregnancy, (ii) breastfeeding, (iii) risk of infectious endocarditis, (iii) use of mouthwash regularly (more than once a week), (iv) inability to comply with protocol, (v) lack of written agreement, (vi) unable to answer questions, and (vii) uncooperative patients.During the inclusion visit, demographic data  and clinical characteristics  were collected from the subjects. Reporting of the nature of the symptoms was recorded at the time of the viral load measurement. An electronic medical record (e-CRF) permitted to record all these informations.At 9 a.m., each subject collected one salivary sample with an accredited health care professional using the Saliva Collection System kit . Only one sample was collected from each adult. Firstly, with the saliva extraction solution, the subject rinsed the oral cavity for 2 min. Secondly, the subject spit into a saliva-collection beaker, without coughing or scraping to clear the throat, to collect the non-stimulated and pure saliva (between 1 and 3.5 mL). Finally, the saliva sample transferred to a sterile tube and stored at 4°C until analysis.RNA was extracted from 200 μL of saliva sample using the NucliSens easyMAG instrument , according to the manufacturer’s guidelines. The RNA was eluted in 50 μL of water and used as a template for real-time (rt) RT-PCR.Real-time RT-PCR assays were performed with the Invitrogen Superscript™ III Platinum One-Step qRT-PCR system . The mix was composed of 5 μL of extracted RNA, 1 μL of Superscript III RT/Platinum Taq Mix, 12.5 μL of 2X reaction buffer, 0.4 μL of a 50 mM magnesium sulfate solution, 1 μL of RdRp-IP2 forward primer (0.4 μM), 1 μL of RdRp-IP2 reverse primer (0.4 μM), 1 μL of RdRp-IP4 forward primer (0.4 μM), 1 μL of RdRp-IP4 reverse primer (0.4 μM). The primer and probe sequences correspond to the RdRp-IP2 and the RdRp-IP4 assays designed at The Institut Pasteur to target a section of the RdRp gene (nt 12621-12727 and 14010-14116 positions) based on the sequences of SARS-CoV-2 (NC_004718) made available on the Global Initiative on Sharing All Influenza Data data-base on 11 January 2020 . These p2 to 106 copies per μL. The SARS-CoV-2 viral load in saliva was calculated as the number of RNA copies per mL of saliva.The assays were performed on a Quant Studio 5  with the following program: 55°C for 10 min (reverse transcription), followed by 95°C for 2 min, and then 45 cycles of 95°C for 3 s and 58°C for 30 s. Each run included three negatives’ samples bracketing unknown samples during RNA extraction, two positive controls, and one negative amplification control. When a sample was positive for RdRp-IP4, the quantification of the number of RNA copies was performed according to a scale ranging from 10p < 0.05.SPSS Windows 20.0  was used for the descriptive statistics median values and interquartile range (IQR) and mean values with SD for the quantitative variables and percentages for categorical variables. One-way ANOVA was used to compare the differences of median between groups in a univariate analysis. Binary logistic regression analysis was used to model the relationship between viral load values as the dependent variable and the other parameters were entered individually as independent variables (adjusted for age and gender). The detailed statistical methods are indicated in the table footnotes. All data were considered statistically significant when The sex, the age and the clinical assessments of the study group are summarized in 10 copies/mL . The first quartile (Q1) corresponded to a viral load starting at 2.95 log10 copies/mL, whereas the second (Q2) corresponded to a viral load starting at 4.12 log10 copies/mL, and the third (Q3) corresponded to a viral load starting at 5.16 log10 copies/mL.Median initial viral load was 4.12 log10 copies/mL (18–34 years) to 4.31 log10 copies/mL (55–77 years). Salivary viral load was not associated with sex. The presence of co-morbidity or not, did not significantly modify the SARS-CoV-2 salivary viral load. Same observation was done for the time between RT-PCR and symptom onset, the delay from symptom onset to saliva sampling and for the number of symptoms per participants. Patients with symptoms demonstrated a median initial viral load of 4.12 log10 copies/mL , while the no-symptoms patients had indices of 4.01 log10 copies/mL .The SARS-CoV-2 salivary viral load according to demographics and clinical characteristics of participants are described in The analysis of the mean difference of SARS-CoV-2 salivary load was reported in 10 copies/mL [IQR −0.34–0.10] was observed. First, an increase 0.26 log10 copies/mL [IQR −0.67–1.18] was observed at 3–5 days compared with 0–2 days. Then, a decrease of −0.39 log10 copies/mL [IQR −1.70–0.92] was observed at 6–8 days.Concerning the time between positive RT-PCR and symptom onset, a non-significant decrease of −0.12 log10 copies/mL [IQR 0.56–5.75] and patients with symptoms   . The pat32–5.59] .P = 0.004 and P = 0.043, respectively)  .10 copies/mL (IQR 2.95–5.16 log10 copies/mL), 27/159 patients (16.98%) had no charge. Between no-symptoms patients with an initially salivary SARS and patients with symptoms, there was no significant median difference in the viral load. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.The present study was designed to explore the quantification of SARS-CoV-2 in pure saliva of adults with asymptomatic to mild COVID-19. This robust multicenter observational trial, including 160 individuals, is the first, to our knowledge, to target this specific population—mean age of patients was 43.62 ± 15.56 years- and to describe the sociodemographic and clinical characteristics of the salivary viral load. The load SARS-CoV-2 value of the whole sample ranged from 0.00 to 10.19 with a median of 4.12 logde novo and a cellular fraction from infected and shed oral mucosa, little is known about the correlation between viral load and age, gender, symptoms, and comorbidity. The first study quantifying the salivary load of SARS-CoV-2 included 12 hospitalized patients with laboratory-confirmed COVID-19, with a median age of 62.5 years. SARS-CoV-2 was detected in 91.7% of throat saliva samples from COVID-19 patients, and the number was as high as 1–2 × 108 infectious copies per milliliter . Since the beginning of the pandemic, there has been controversy about the infectivity of asymptomatic patients. With the aforementioned limitation on the robustness of existing studies which are based on considerably smaller data sets, most studies demonstrate more rapid viral clearance in asymptomatic individuals than in symptomatic ones  in 30–60% of infected individuals, especially in young adults and adults . Asymptotic ones . A largetic ones . It is rtic ones . Additiotic ones . The ambtic ones .p = 0.04  and not the Delta variant (B.1.617.2) which is now detected in the majority of cases. Thus, it is likely that the results would be different if the study were conducted now since the delta variant has different characteristics .Secondly, we also clearly explained the ambiguity surrounding asymptomatic vs. presymptomatic status. We describe them as suggestive, not conclusive. While measuring viral load can be useful in clinical practice, a positive RT-qPCR result does not necessarily mean that the person is still infectious or still has significant disease. The RNA could be from a non-viable virus and/or the amount of live virus could be too low to allow transmission . Contactpost hoc studies have revealed the importance of SARS-CoV-2 corona virus 2 transmission from both asymptomatic and mildly symptomatic cases, the virologic basis for their infectivity remains largely unquantified (Strengths of our study is that, although antified . DespiteOur results raise the possibility that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission. This result may have public health implications if enhanced saliva samples are used for asymptomatic screening. Considering oral SARS-CoV-2 infection and the ease of saliva for transmission, it remains critical to further understanding of the dominant modes of viral spread across the spectrum of asymptomatic, pre-symptomatic and symptomatic individuals.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Committee for the Protection of Persons South Mediterranean III, France. The patients/participants provided their written informed consent to participate in this study.FC, DB, and CD proposed the original study idea and designed the trial and study protocol. DB and FC contributed to the data interpretation and wrote the first draft of the manuscript. FC, CD, and HP verified the data. EG, AE, ML, and JD were responsible for the site work including the recruitment, follow up and data collection. HP monitored the trial. DB did the main analysis. CD, EG, and JD contributed to the revision of the manuscript. All authors reviewed and accepted the manuscript before submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.